Is substrate choice an overlooked variable in ecotoxicology experiments?

It is crucial to understand the effects caused by experimental parameters such as temperature, light, and food type on lab and field-based ecotoxicology experiments, as these variables, and combinations thereof, can affect results. The type of substrate used in exposure experiments, however, is generally assumed to have no effect. This may not always be correct. The metabolic changes in the freshwater crustacean, Austrochiltonia subtenuis exposed to copper, using three common substrates, gauze; toilet paper; and cellulose were investigated. Substrate alone did not affect survival, but each substrate elicited a different metabolic response and adult and juvenile amphipods had different substrate preferences. Several classes of metabolites were shown to change in response to different substrates and toxicant. These included disaccharides, monosaccharides, fatty acids, and tricarboxylic acid cycle intermediates. The results illustrate that metabolomic responses can differ in response to experimental factors that were previously thought not to be significant. In fact, our data indicate that substrate should be viewed as an experimental factor as important to control for as more well-known confounders such as temperature or food, thus challenging the current paradigm. Assuming substrate type has no effect on the experiment could potentially lead to errors in contaminant toxicity assessments. We propose that ideal good practise would be that all experimental factors should be evaluated for their potential influence on metabolomic profiles prior to contaminant response experiments being undertaken.

Using metabolomics to assess the sub-lethal effects of zinc and boscalid on an estuarine polychaete worm over time.

INTRODUCTION: Zinc is a heavy metal commonly detected in urban estuaries around Australia. Boscalid is a fungicide found in estuaries, both in water and sediment, it enters the system predominantly through agricultural run-off. Zinc is persistent while boscalid breaks down, with a half-life of 108 days. Both contaminants are widely distributed and their effects on ecosystems are not well understood. OBJECTIVES: The aim of this study was to determine the metabolite changes in Simplisetia aequisetis (an estuarine polychaete) following laboratory exposure to a sub-lethal concentration of zinc or boscalid over a 2-week period. METHODS: Individuals were collected at six time points over a 2-week period. Whole polychaete metabolites were extracted and quantified using a multi-platform approach. Polar metabolites were detected using a semi-targeted GC-MS analysis and amine containing compounds were analysed using a targeted LC-MS analysis. Total lipid energy content was also analysed for Simplisetia aequisetis. RESULTS: The pathways that responded to zinc and boscalid exposure were alanine, aspartate and glutamate metabolism (AAG); glycine, serine and threonine metabolism (GST) and metabolites associated with the tricarboxylic acid cycle (TCA). Results showed that changes in total abundance of some metabolites could be detected as early as 24-h exposure. Changes were detected in the metabolites before commonly used total lipid energy assays identified effects. CONCLUSION: A multi-platform approach provided a holistic overview of the metabolomic response to contaminants in polychaetes. This approach shows promise to be used in biomonitoring programs to provide early diagnostic indicators of contamination and exposure.

A Meta-Analysis Evaluating the Relationship between Aquatic Contaminants and Chironomid Larval Deformities in Laboratory Studies.

Chironomid larval deformities have been widely investigated as an aquatic pollution toxicity end point. Field chironomid surveys often show a spatial association between contaminants and deformities, suggesting contaminants cause deformities. However, over 40 years of laboratory assays have not been able to confirm this causality. We therefore conducted a review of the literature and meta-analysis, in order to (A) assess whether trends across assays indicated dose-response effects, (B) characterize the consistency of results, and (C) investigate whether experimental issues and publication bias were contributing to inconsistency and/or reducing confidence in results. The experimental issues we investigated were extraneous nonchemical laboratory stressors (which may mask or interact with chemical effects), and mortality (which can confound deformity results). Our meta-analysis of the most commonly tested chemicals suggested dose-response effects for copper, but not lead or zinc. However, we also found substantial inconsistency across studies. Both mortality and extraneous stressors were potentially contributing to this inconsistency, reducing confidence in most published data. We observed no evidence of publication bias. We conclude that any causal link between contaminants and deformities remains uncertain, and suggest improved experimental and data reporting procedures to better assess this relationship.

Comparing the impacts of sediment-bound bifenthrin on aquatic macroinvertebrates in laboratory bioassays and field microcosms.

We conducted two laboratory bioassays and two field microcosm exposures with bifenthrin (a synthetic pyrethroid) in order to evaluate the capacity of single-species laboratory bioassays to predict lethal and sublethal impacts on aquatic invertebrates in microcosms. For the laboratory species, Chironomus tepperi, larval survival was reduced by 24% at 53.66µg/g OC, while adult emergence was reduced at concentrations of 33.33µg/g OC and higher, with a 61% decrease at 77.78µg/g OC and no emergence at 126.67µg/g OC. The abundance of several other microcosm taxa was reduced in the microcosms at a similar concentration range (33.33µg/g OC and above), however there was no impact on the abundance of the congeneric species, Chironomus oppositus. The differences in impacts between test systems were potentially due to both differing species sensitivity and the interaction of ambient temperature with bifenthrin toxicity. Bifenthrin also was associated with early emergence of Chironomus sp. in both test systems, at concentrations of 10µg/g OC and higher (laboratory) and 43.90µg/g OC (microcosm), and with a significant decrease in the proportion of C. oppositus males in a microcosm. These findings indicate that while laboratory bioassays accurately predict many impacts in the field, there are some limitations to the predictive capacity of these tests.

The Parthenogenetic Cosmopolitan Chironomid, Paratanytarsus grimmii, as a New Standard Test Species for Ecotoxicology: Culturing Methodology and Sensitivity to Aqueous Pollutants.

Chironomids from the genus Chironomus are widely used in laboratory ecotoxicology, but are prone to inbreeding depression, which can compromise test results. The standard Chironomus test species (C. riparius, C. dilutus and C. yoshimatsui) are also not cosmopolitan, making it difficult to compare results between geographic regions. In contrast, the chironomid Paratanytarsus grimmii is cosmopolitan, and not susceptible to inbreeding depression because it reproduces asexually by apomictic parthenogenesis. However, there is no standardised culturing methodology for P. grimmii, and a lack of acute toxicity data for common pollutants (metals and pesticides). In this study, we developed a reliable culturing methodology for P. grimmii. We also determined 24-h first instar LC50s for the metals Cu, Pb, Zn, Cd and the insecticide imidacloprid. By developing this culturing methodology and generating the first acute metal and imidacloprid LC50s for P. grimmii, we provide a basis for using P. grimmii in routine ecotoxicological testing.

Exposure to PFAS contaminated urban wetland water causes similar metabolic alterations to laboratory-based exposures in the freshwater amphipod Austrochiltonia subtenuis.

Assessing the harm caused by pollutants in urban ecosystems remains a significant challenge. Traditional ecotoxicological endpoints are often not sensitive enough to detect the effects of toxicants at environmentally relevant concentrations (≤ng/L). A potential solution is using molecular biology methods to look at small biochemical changes caused by exposure to ng/L concentrations of contaminants. This has been tested in the lab but not conclusively demonstrated in the field. We exposed the freshwater amphipod (Austrochiltonia subtenuis) to water from an urban wetland containing known concentrations of per-and polyfluoroalkyl substances (as well as very low concentrations of pesticides) for 14 days and analyzed their metabolite profiles. Mannose, Myo-inositol, and Isopropyl propionate were found to change in PFAS exposed amphipods, a similar response to that previously observed in laboratory exposures to the same PFAS, but not pesticides. The results give a better understanding of PFAS toxicity at environmentally relevant concentrations and conditions.

Changes in metabolic profiles of amphipods Allorchestes compressa after acute exposures to copper, pyrene, and their mixtures.

Amphipods are ideal indicators for biomonitoring and ecotoxicological studies of environmental contaminants because they are extensively distributed in aquatic environments, are easy to collect and are important in nutrient cycling. Marine amphipods (Allorchestes compressa) were exposed to two concentrations of copper and pyrene, and their mixtures, for 24 and 48 h. Changes in polar metabolites were assessed using Gas Chromatography Mass Spectrometry (GC-MS)-based untargeted metabolomics. Generally, limited metabolite changes were observed for copper and pyrene single exposures (eight and two significant metabolites, respectively), while 28 metabolites had changed following exposures to mixtures. Furthermore, changes were mainly observed after 24 h but had seemingly returned to control levels after 48 h. Multiple types of metabolites were affected including amino acids, Tricarboxylic acid (TCA) cycle intermediates, sugars, fatty acids, and hormones. This study highlights the sensitivity of metabolomics in assessing the impacts of low concentrations of chemicals compared to traditional ecotoxicological endpoints.

A review of omics-based PFAS exposure studies reveals common biochemical response pathways.

Per and Polyfluoroalkyl Substances (PFAS) are a diverse group of man-made chemicals with a range of industrial applications and which are widespread in the environment. They are structurally diverse but comprise a common chemical feature of at least one (though usually more) perfluorocarbon moiety (-CnF2n-) attached to a functional group such as a carboxylic or sulphonic acid. The strength of the Carbon-Fluorine bond means the compounds do not break down easily and can thus bioaccumulate. PFAS are of high concern to regulators and the public due to their potential toxicity and high persistence. At high exposure levels, PFAS have been implicated in a range of harmful effects on human and environmental health, particularly problems in/with development, cholesterol and endocrine disruption, immune system function, and oncogenesis. However, most environmental toxicology studies use far higher levels of PFAS than are generally found in the environment. Additionally, since the type of exposure, the PFAS used, and the organisms tested all vary between studies, so do the results. Traditional ecotoxicology studies may thus not identify PFAS effects at environmentally relevant exposures. Here we conduct a review of omics-based PFAS exposure studies using laboratory ecotoxicological methodologies and environmentally relevant exposure levels and show that common biochemical response pathways are identified in multiple studies. A major pathway identified was the pentose phosphate shunt pathway. Such molecular markers of sublethal PFAS exposure will greatly benefit accurate and effective risk assessments to ensure that new PFAS regulations can consider the full effects of PFAS exposure on environmental and human health receptors.

Exposure to Environmentally Relevant Levels of PFAS Causes Metabolic Changes in the Freshwater Amphipod Austrochiltonia subtenuis.

Per and polyfluoroalkyl substances (PFAS) are of concern to environmental regulators due to their widespread occurrence, persistence and reported toxicity. However, little data exist on the effects of PFAS at environmentally relevant concentrations. The development of molecular markers for PFAS exposure would therefore be useful to better understand the environmental risks of these compounds. In this study, we assessed if such markers could be developed using Gas Chromatography-Mass Spectrometry-based metabolomics. We exposed the freshwater amphipod Austrochiltonia subtenuis to a range of environmentally relevant concentrations of perfluoro-octane sulfonic acid (PFOS), hexafluoropropylene oxide dimer acid (GenX) and perfluorohexanesulphonic acid (PFHxS) for 7 days at five concentrations. A metabolic response was detected in all concentrations and treatments even though the survival rates only differed significantly at the highest exposure levels. The metabolic response differed between compounds but all three PFAS induced changes in the levels of amino acids, fatty acids, and cholesterol, in line with the literature. PFOS was found to bioaccumulate. Both GenX and PFHxS were eliminated from the amphipods, but PFHxS was eliminated at a slower rate than GenX. This information improves our understanding of the sublethal effects of PFAS as well as their environmental fate and behaviour.

What are the effects of PFAS exposure at environmentally relevant concentrations?

The group of synthetic chemicals known as poly and per-fluoroalkyl substances (PFAS) are currently of high concern to environmental regulators and the public due to their widespread occurrence, resistance to degradation and reported toxicity. However, little data exists on the effects of exposure to PFAS at environmentally relevant concentrations and this hampers the effective management of these compounds. This paper reviews current research on the occurrence and ecotoxicology of PFAS at environmentally relevant doses to assess their potential biological impacts. Hazard Quotient (HQ) analysis was undertaken as part of this assessment. Most PFAS detected in the environment were found to have a HQ risk value of <1 meaning their reported concentrations are below their predicted no effect concentration. This indicates many reported toxic effects of PFAS are, theoretically, unlikely to occur outside the laboratory. However, lack of information on new PFAS as well as their precursors and degradation products, coupled with lack of knowledge of their mixture toxicity means our understanding of the risks of PFAS is incomplete, especially in regard to sub-lethal and/or chronic effects. It is proposed that the development of molecular markers for PFAS exposure are needed to aid in the development of environmental PFAS regulations that are effective in fully protecting the environment.

What types of enzyme activities are useful biomarkers of bifenthrin exposure on Chironomus sp. (Diptera, Chironomidae) larvae under laboratory and field-based microcosm conditions?

Bifenthrin is a second generation synthetic pyrethroid insecticide that is widely used in Australia and worldwide. It is frequently found in urban freshwater sediments at concentrations likely to impact biota as it is highly toxic to fish and macroinvertebrates, such as chironomids. Our main goal was to evaluate if oxidative stress and hydrolase enzymes are useful biomarkers of effect of synthetic pyrethroids exposure under different scenarios. Chironomus tepperi larvae (5 days old) were exposed to sub-lethal sediment concentrations of bifenthrin for 5 days under controlled laboratory conditions. A field-based microcosm exposure with bifenthrin-spiked sediments (using the same concentrations as the laboratory exposure) was carried out at a clean field site for four weeks to allow for colonization and development of resident chironomid larvae. At the end of both experiments, Chironomus larvae (C. tepperi in the laboratory exposures and C. oppositus in the microcosm exposures) were collected and oxidative stress enzymes (Glutathione-s-Transferase, Glutathione Reductase and Glutathione Peroxidase) and hydrolase enzymes (Acetylcholinesterase and Carboxylesterase) were measured. Only the Glutathione Peroxidase activity was significantly impacted in larvae from the laboratory exposure. On the contrary, significant changes were observed in all the measured enzymes from the field-based microcosm exposure. This is likely because exposure was throughout the whole life cycle, from egg mass to fourth instar, showing a more realistic exposure scenario. Furthermore, this is the first time that changes in oxidative stress and hydrolase enzymes have been shown to occur in Australian non-biting midges exposed under field-based microcosm conditions. Thus, this study demonstrated the usefulness of these enzymes as biomarkers of effect following bifenthrin exposure in microcosms. It also highlights the importance of using a range of different biochemical endpoints to get a more holistic understanding of pesticide effects and the pathways involved.

Metabolomics Provide Sensitive Insights into the Impacts of Low Level Environmental Contamination on Fish Health-A Pilot Study.

This exploratory study aims to investigate the health of sand flathead (Platycephalus bassensis) sampled from five sites in Port Phillip Bay, Australia using gas chromatography-mass spectrometry (GC-MS) metabolomics approaches. Three of the sites were the recipients of industrial, agricultural, and urban run-off and were considered urban sites, while the remaining two sites were remote from contaminant inputs, and hence classed as rural sites. Morphological parameters as well as polar and free fatty acid metabolites were used to investigate inter-site differences in fish health. Significant differences in liver somatic index (LSI) and metabolite abundance were observed between the urban and rural sites. Differences included higher LSI, an increased abundance of amino acids and energy metabolites, and reduced abundance of free fatty acids at the urban sites compared to the rural sites. These differences might be related to the additional energy requirements needed to cope with low-level contaminant exposure through energy demanding processes such as detoxification and antioxidant responses as well as differences in diet between the sites. In this study, we demonstrate that metabolomics approaches can offer a greater level of sensitivity compared to traditional parameters such as physiological parameters or biochemical markers of fish health, most of which showed no or little inter-site differences in the present study. Moreover, the metabolite responses are more informative than traditional biomarkers in terms of biological significance as disturbances in specific metabolic pathways can be identified.

Combined chemical (fluoranthene) and drought effects on Lumbricus rubellus demonstrate the applicability of the independent action model for multiple stressor assessment.

The combined effect of a chemical (fluoranthene) and a nonchemical stress (reduced soil moisture content) to the widely distributed earthworm Lumbricus rubellus were investigated in a laboratory study. Neither fluoranthene (up to 500 microg/g) nor low soil moisture (15% below optimal) had a significant effect on the survival of the exposed worms, but a significant effect on reproduction (cocoon production rate) was found for both stressors (p < 0.001 in both cases). The response of cocoon production to each stressor could be well described by a logistic model; this suggested that the joint effects may be applicable to description using the independent action (IA) model that is widely used in pharmacology and chemical mixture risk assessment. Fitting of the IA model provided a good description of the combined stressor data (accounting for 53.7% of total variation) and was the most parsimonious model describing joint effect (i.e., the description of the data was not improved by addition of further parameters accounting for synergism or antagonism). Thus, the independent action of the two responses was further supported by measurement of internal fluoranthene exposure. The chemical activity of fluoranthene in worm tissue was correlated only with soil fluoranthene concentration and not with soil moisture content. Taken together these results suggest that the IA model can help interpret the joint effects of chemical and nonchemical stressors. Such analyses should, however, be done with caution since the literature data set suggests that there may be cases where interactions between stressors result in joint effects that differ significantly from IA predictions.

A Re-Evaluation of Chironomid Deformities as an Environmental Stress Response: Avoiding Survivorship Bias and Testing Noncontaminant Biological Factors.

Larval deformities have been observed in chironomids, and are thought to be associated with aquatic contaminant exposure. However, in laboratory assays, deformities have not been linked with contaminants in the absence of potential confounding variables including mortality, which introduces a survivorship bias. There is also a paucity of data on noncontaminant causes. In addition, power analyses are rarely undertaken, meaning that effect sizes detectable are usually uncertain. We therefore aimed to clarify factors associated with deformities, by running survivorship bias-free (i.e., sublethal) assays, assessing contaminant (copper and imidacloprid) and noncontaminant (malnutrition) stressors, and considering natural biological (metamorphosis) factors in Chironomus tepperi. We included a posteriori power analyses for all tests. Our assays found no significant association between tested factors and deformity rate. Power analyses indicated that the stressor experiment had moderate power to detect deformity effects. The metamorphosis assay had relatively lower power (due to an unexpectedly high control deformity rate), highlighting the importance of power tests in these types of evaluations. These results, in conjunction with others recently published, raise doubts as to the causal effects of environmental stressors on deformity incidence. By avoiding survivorship bias, and by testing noncontaminant factors and statistical power, we present a more holistic methodology, to resolve ongoing uncertainty in this area. We also discuss possible future directions for chironomid deformity research, and concerns regarding survivorship bias in ecotoxicology. Environ Toxicol Chem 2019;38:1658-1667. © 2019 SETAC.

Metabolite Changes in an Estuarine Annelid Following Sublethal Exposure to a Mixture of Zinc and Boscalid.

Environmental pollutants such as heavy metals and fungicides pose a serious threat to waterways worldwide. Toxicological assessment of such contaminants is usually conducted using single compound exposures, as it is challenging to understand the effect of mixtures on biota using standard ecotoxicological methods; whereas complex chemical mixtures are more probable in ecosystems. This study exposed Simplisetia aequisetis (an estuarine annelid) to sublethal concentrations of a metal (zinc) and a fungicide (boscalid), both singly and as a mixture, for two weeks. Metabolomic analysis via gas and liquid chromatography-mass spectrometry was used to measure the stress response(s) of the organism following exposure. A total of 75 metabolites, including compounds contributing to the tricarboxylic acid cycle, the urea cycle, and a number of other pathways, were identified and quantified. The multiplatform approach identified distinct metabolomic responses to each compound that differed depending on whether the substance was presented singly or as a mixture, indicating a possible antagonistic effect. The study demonstrates that metabolomics is able to elucidate the effects and mode of action of contaminants and can identify possible outcomes faster than standard ecotoxicological endpoints, such as growth and reproduction. Metabolomics therefore has a possible future role in biomonitoring and ecosystem health assessments.

Second generation anticoagulant rodenticides in tawny owls (Strix aluco) from Great Britain.

Secondary exposure of vertebrate predators to second generation anticoagulant rodenticides (SGARs) is widespread in Britain. Tawny owl (Strix aluco) populations in the UK are thought to have declined since the 1970s, when SGARs were first introduced, and these compounds may have contributed to any decline in owl numbers. Our aims were to conduct the first systematic survey of SGAR exposure in tawny owls and ascertain whether there had been a change in the proportion of exposed birds that was concurrent with the decline in the population. Liver difenacoum, bromadiolone, flocoumafen and brodifacoum concentrations in British tawny owls from two periods (1990-1993 and 2003-2005) were quantified. In total, some 20% of birds contained detectable residues of one or more SGAR. The extent of exposure (% of birds exposed, magnitude of residues) to different SGARs did not change consistently between time periods. Of the raptors analysed to date in Britain, tawny owls had the lowest proportion of individuals that contained detectable liver residues and so appear to be the least vulnerable to exposure and/or assimilation of SGARs. We found no clear evidence to implicate SGARs as a major factor affecting tawny owl numbers in Britain between 1990 and 2005.

Structural and functional measures of marine microbial communities: An experiment to assess implications for oil spill management.

Microbial communities are ecologically important in aquatic environments and impacts on microbes have the potential to affect a number of functional processes. We have amended seawater with a crude oil and assessed changes in species composition as well as a measure of functional diversity (the ability of the community to utilise different carbon sources) and the community level metabolic signature. We found that there was a degree of functional redundancy in the community we tested. Oiled assemblages became less diverse and more dominated by specialist hydrocarbon degraders, carbon source utilisation increased initially but there was no change in metabolic signature in this small scale laboratory experiment. This study supports the decision framework around management of oil spills. This package of methods has the potential to be used in the testing and selection of new dispersants for use in oil spill response.

Effects of two commonly used fungicides on the amphipod Austrochiltonia subtenuis.

Fungicides are used widely in agriculture and have been detected in adjacent rivers and wetlands. However, relatively little is known about the potential effects of fungicides on aquatic organisms. The present study investigated the effects of 2 commonly used fungicides, the boscalid fungicide Filan® and the myclobutanil fungicide Systhane™ 400 WP, on life history traits (survival, growth, and reproduction) and energy reserves (lipid, protein, and glycogen content) of the amphipod Austrochiltonia subtenuis under laboratory conditions, at concentrations detected in aquatic environments. Amphipods were exposed to 3 concentrations of Filan (1 µg active ingredient [a.i.]/L, 10 µg a.i./L, and 40 µg a.i./L) and Systhane (0.3 µg a.i./L, 3 µg a.i./L, and 30 µg a.i./L) over 56 d. Both fungicides had similar effects on the amphipod at the organism level. Reproduction was the most sensitive endpoint, with offspring produced in controls but none produced in any of the fungicide treatments, and total numbers of gravid females in all fungicide treatments were reduced by up to 95%. Female amphipods were more sensitive than males in terms of growth. Systhane had significant effects on survival at all concentrations, whereas significant effects of Filan on survival were observed only at 10 µg a.i./L and 40 µg a.i./L. The effects of fungicides on energy reserves of the female amphipod were different. Filan significantly reduced amphipod protein content, whereas Systhane significantly reduced the lipid content. The present study demonstrates wide-ranging effects of 2 common fungicides on an ecologically important species that has a key role in trophic transfer and nutrient recycling in aquatic environments. These results emphasize the importance of considering the long-term effects of fungicides in the risk assessment of aquatic ecosystems. Environ Toxicol Chem 2017;36:720-726. © 2016 SETAC.

Toxicological effects of fungicide mixtures on the amphipod Austrochiltonia subtenuis.

Approaches to assess the toxicity of mixtures often use predictive models with acute mortality as an endpoint at relatively high concentrations. However, these approaches do not reflect realistic situations where organisms could be exposed to chemical mixtures over long periods at low concentrations at which no significant mortalities occur. The present study investigated chronic effects of 2 common fungicides, Filan® (active ingredient [a.i]) boscalid) and Systhane™ (a.i. myclobutanil), on the amphipod Austrochiltonia subtenuis at environmentally relevant concentrations under laboratory conditions. Sexually mature amphipods were exposed singly and in combination to Filan (1, 10, and 40 µg a.i./L) and Systhane (3 µg a.i./L) over 28 d. Survival, growth, a wide range of reproduction endpoints, and glutathione-S-transferase (GST) activity were measured at the end of the experiment. Both fungicides had significant independent effects on male growth, sex ratio, and juvenile size. Filan mainly affected female growth and the number of embryos per gravid female, whereas Systhane mainly affected the time for females to become gravid. The combined effects of these fungicides on numbers of gravid females and juveniles were antagonistic, causing a 61% reduction in the number of gravid females and a 77% reduction in the number of juveniles produced at the highest concentrations (40 µg a.i./L of boscalid and 3 µg a.i./L of myclobutanil) compared with the controls. There were no significant effects on survival or GST activity. The present study demonstrated that the effects of mixtures were endpoint dependent and that using a variety of endpoints should be considered for a comprehensive understanding of mixture effects. Also, chronic studies are more informative than acute studies for environmentally relevant fungicide concentrations. Environ Toxicol Chem 2017;36:2651-2659. © 2017 SETAC.

Municipal wastewater effluent licensing: A global perspective and recommendations for best practice.

Advances in wastewater treatment have greatly improved the quality of municipal wastewater effluents in many parts of the world, but despite this, treated wastewaters can still pose a risk to the environment. Licensing plays a crucial role in the regulation of municipal wastewater effluents by setting standards or limits designed to protect the economic, environmental and societal values of waterbodies. Traditionally these standards have focused on physical and chemical water quality parameters within the discharge itself, however these approaches do not adequately account for emerging contaminants, potential effects of chemical mixtures, or variations in the sensitivity and resilience of receiving environments. In this review we focus on a number of industrialised countries and their approach to licensing. We consider how we can ensure licensing is effective, particularly when considering the rapid changes in our understanding of the impacts of discharges, the technical advances in our ability to detect chemicals at low concentrations and the progress in wastewater treatment technology. In order to meet the challenges required to protect the values of our waterways, licensing of effluents will need to ensure that there is no disconnect between the core values to be protected and the monitoring system designed to scrutinise performance of the WWTP. In many cases this may mean an expansion in the monitoring approaches used for both the effluent itself and the receiving waterbody.